Our goal is to understand how Ca 2+, acting as a second messenger, regulates cellular processes. It is now well established that in response to external signals changes in intracellular Ca 2+are recognized by regulatory Ca 2+-binding proteins which act as sensors and modulators of calcium transients. Calmodulin (CaM) is a unique member of this protein family because of its ability to activate a large number of enzymes. To understand how CaM can activate so many proteins we used as a model system the regulation of the protein phosphatase, calcineurin, by Ca 2+ /CaM. The identification of calcineurin as the target of the immunosuppresive drugs, cyclosporin A and tacrolimus (FK506) by S. Schreiber revealed the key role of this enzyme in T cell activation and led to the identification of calcineurin as a major player in cellular processes as diverse as cell growth and differentiation, programmed cell death, embryogenesis , and neuronal functions. Thus, it is no surprise that alteration of calcineurin activity has been implicated in the pathogenesis of an increasing number of diseases. We have focused our attention to the complex regulation of this key enzyme in Ca 2+ signaling. Our major goal is to crystallize the b-isoform of calcineurin depleted of Ca 2+ and complexed with CaM in order to understand, at the molecular level, why Ca 2+ binding to two Ca 2+ -regulated proteins, calcineurin B (CnB) an integral subunit of the enzyme and CaM,is needed to activate calcineurin. The b-isoform was selected because of its broad distribution as opposed to the predominantly neural distribution of a-isoform. The b-isoform of the catalytic subunit of calcineurin (CnA) expressed in E. Coli, always found in inclusion bodies, was solubilized in denaturing solvents and refolded into an active enzyme that requires CnB for activity and stimulation by CaM. Refolded CnA is 90% pure without further purification. It is being used to understand why binding of Ca 2+ to CnB is required for the interaction of calcineurin with CaM, endogenous inhibitors and immunosuppressive drugs. Coexpression of CnA with CnB yields a soluble enzyme that can be purified to homogeneity (H. Ren) and will be used in collaboration with Dr. Xia (Laboratory of Cell Biology, NCI) to crystallize the inactive and CaM activated forms of calcineurin. The ultimate goal is to obtain the information needed to design more selective and less toxic drugs We previously showed, in collaboration with Jay Zweier at Johns Hopkins University, that reactive oxygen species play a role in the Ca 2+/CaM-dependent inactivation of calcineurin prevented by superoxide dismutase (SOD). This reversible inactivation provides a mechanism for coupling Ca 2+ signaling to the redox potential of the cell. During the past year, in collaboration with Dr. Charles Krieger we have analyzed calcineurin activity in brain tissue extracts of mice overexpressing a mutant SOD which has been implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS), to test the possibility that calcineurin is involved in the development of ALS.